The present invention relates to thermostable analyte sensors. More particularly, the invention relates to electrochemical sensors for the measurement of bioanalytes such as glucose and lactate. The inventive sensors include a thermostable peroxidase, such as soybean peroxidase, disposed on an electrode in a redox-compound-containing film. The sensor further includes a hydrogen peroxide-producing enzyme. The inventive sensors, for example in the measurement of blood glucose or lactate, operate for five days or more at 37xc2x0 C., losing less than 10% of their sensitivity during continuous or intermittent operation.
The assay of biochemicals, such as glucose and lactate, is important in medicine, biotechnology, and food processing (dairy and wine). Monitoring of glucose concentrations in fluids of the human body is of particular relevance to diabetes management. Monitoring of lactate in fluids of the human body is of relevance to diagnosis of trauma, of myocardial infarction, congestive heart failure, pulmonary edema, septicemia, hemorrhage, and others. Blood lactate levels above 7-8 mM are indicative of a fatal outcome. Bedside analyzers of lactate are useful in determining the response of patients to treatment, while in accidents and battle, they are useful in triage. Glucose assays are common in clinical practice and are applied in the diagnosis of Diabetes Mellitus and its management. Continuously or intermittently operating glucose sensors, including sensors implanted in the human body, are sought for the management of Type I diabetes, for example, for warning of imminent or actual hypoglycemia and its avoidance. Hypoglycemia can be fatal. For maintenance of diabetic patients at or near normal blood glucose levels, frequent or continuous monitoring of glucose is needed. Today, most Type I diabetic patients maintain their blood glucose at higher than normal levels, so as to reduce risk of fatal hypoglycemia. This is undesirable, as maintenance of higher than normal blood glucose levels has been shown to be a leading cause of blindness, kidney failure, neuropathy, and other complications of diabetes. It would, therefore, be useful to provide a glucose sensor that operates continuously or intermittently for a prolonged period of time to measure glucose in body fluid at 37xc2x0 C. without substantial loss of sensitivity.
The present invention discloses material, structures, and methods enabling continuous operation of electrochemical sensors, for example, measuring glucose for more than one week or measuring lactate for more than 100 hours, with less than 10% loss in sensitivity.
Novel electrochemical sensors are presented, some of which are capable of operating at 37xc2x0 C. continuously or intermittently, measuring biochemicals in body fluids with less than 10% loss of sensitivity in more than 100 hours of operation are described herein. The inventive sensors are relatively insensitive to electrooxidizable interferants, including ascorbate and acetaminophen.
The sensors of the invention include at least two enzymes, a thermostable peroxidase, such as soybean peroxidase, and a peroxidase-generating enzyme. In a preferred embodiment, redox centers of a thermostable peroxidase are electroreduced by electrons transported from a working electrode through a redox hydrogel in which the thermostable peroxidase is immobilized, preferably at a potential negative of 0.4 V versus the standard calomel electrode (SCE) and positive of xe2x88x920.15 V (vs SCE). Most preferably, the thermostable peroxidase is coated on the electrode at a potential near 0.0V (vs SCE).
The preferred redox hydrogel comprises at least 20% by weight of water when in contact with a fluid to be assayed, and its redox centers are not leached by the assay fluid at 37xc2x0 C. Non-leachable redox centers are bound to a polymer that forms the hydrogel upon water uptake. Preferably, the binding to the polymer is through covalent, electrostatic/ionic, or coordination bonds.
The redox centers of the peroxide-generating enzyme are preferably electrically insulated from the electrode, from the redox centers of the thermostable peroxidase, and from the redox centers of the redox hydrogel. The electrically insulated, peroxide-generating enzyme catalyzes reaction of a biochemical analyte, e.g., glucose or lactate, or a product of the analyte, with molecular oxygen. In the oxidation reaction, oxygen is reduced to hydrogen peroxide (H2O2).
The hydrogen peroxide-generating enzyme is preferably stabilized in a matrix. The preferred stabilizing matrices are macromolecular and inorganic. The most preferred matrices include silicon atoms, at least 50% of which are covalently linked to neighboring oxygen atoms, which are formed into a three-dimensional, crosslinked network. Such matrices can be made using a sol-gel polymerization process. The stabilizing matrix optionally includes a second polymer, which functions to further stabilize the insulated, peroxide-generating enzyme.
The peroxide-generating enzyme is preferably positioned behind or immobilized in a polymer that is at least tenfold, and preferably at least 100-fold, more permeable to oxygen than is the biochemical analyte to be measured. Examples of such polymers include silicone rubbers, produced by cross-linking a poly(dimethyl siloxane) derivative and cellulose acetate.